Ciliated Cells Of Nasal Mucusa Differentiated From Human Umbilical Cord Blood Mesenchymal Stem Cells

Rationale Many diseases can do harm to nasal mucusa, such as chronic rhinosinusitis, allergic rhinitis, asthma, cystic fibrosis, endoscopic surgery and so on. Recent studies have suggested that both embryonic stem cells and adult bone marrow stem cells can participate in the generation and repair of diseased adult organs, including the lungs. However, the extent of airway epithelial remodeling with adult marrow stem cells is low, and there are no available in vivo data with embryonic stem cells. Human umbilical cord blood contains both hematopoietic and nonhematopoietic stem cells, which is mesenchymal stem cells (MSCs).It is safely and easily obtained immediately after birth and is a rich source. We hypothesized that human umbilical cord blood stem cells might be an effective alternative for regeneration the ciliated cell.Part one Construction of the Culture System for Human Umbilical Cord Blood Mesenchymal Stem CellsObjectives To explore the conditions for isolation, purification and culture of human umbilical cord blood derived mesenchymal stem cells(MSCs) in vitro. To set up a stable culture system to meet the experimental needs.Methods The umbilical cord blood was collected from full term normal and premature deliveries or scheduled for cesarean section, which was agreed by the parturients and their relatives.The specimens were obtained sterilely with preservative-free heparin,and the cord blood mononuclear cells(MNCs) were isolated by lymphocyte separation medium and 6% Hydroxyethyl Starch.Then the MNCs were cultured in the six-well culture plates coated by FBS.After cultured for 7 days, the DMEM(10% FBS) was changed for the first time and remove non-adherent cells. Since then on the culture medium was changed for five days. The adherent cells were observed. The P3 cell were collected and cell antigens (CD13,CD44,CD34,CD45) were detected by flow cytometry. At the same time , the cell cycle analysis was detected.Results MSCs were successfully isolated from umbilical cord blood by lymphocyte separation medium and 6% Hydroxyethyl Starch. The Primary culture was passaged when the cells were grown to 70-80% confluent. The P3 cells had good Uniformity of cell size and uniformity of cell shape. The plastic adherent cells express CD13, CD44, but not or low expression of CD34 and CD45.This results were similar to bone marrow mesenchymal stem cells and Unlike hematopoietic cell. Cell cycle analysis showed that 95.29% P3 cells were in G0/G1 cell cycle. In this study, the mononuclear cells (MNCs) were got form 18 cord blood. But only three cord blood from premature delivery (34-37weeks) and one cord blood from full term fetus (38 weeks) got more MSCs that could be passaged.Conclusions Mononuclear cells were seeded in DMEM culture medium containing 10%FBS and six-well culture plates coated with FBS. MSCs can be cultivated successfully in vitro. The cord blood from premature delivery contained more MSCs.Part two Culture and Identification of Humal Nasal Epithelial CellsObjectives To establish cell culture system of human nasal epithelial air-liquid interface (ALI) cell culture and the classical submerged single layer (SSL) cell culture model,and select the method to induce cilia cell.Methods The specimens of healthy nasal mucusa from surgery for snoring under general anaesthesia were placed into 4。C sterile phosphate-buffered saline(PBS) solution supplemented with gentamicin and fluconazole, washed repeatedly, immersed, cut into pieces, transferred into culture bottles, digested with 0.1% collagenaseⅣover night. On the next day, the samples were removed , pipetted slightly to detach, cells were cultured in six well plates in SSL and transwell polycarbonate membrane insert coated by rat tail tendon collagen type I in ALI.Cell were observed on inverted phase microscopy.HE and PAS staining were observed on light microscopy . Immunofluorescent staining of FOXJ1 andβ-Tubulin were observed on fluorescence microscope.Cells cultured in SSL were selected to observed under the transmission electron microscopy and scanning electron microscopy. The P5 cell were detected chromosome.Results SSL and ALI could find the objective cells. The ciliated cells and goblet cells in SSL were observed. They expressed cytokeratin 14( CK14) , which was a good marker for epithelial cells. They expressed FOXJ1 andβ-Tubulin, which were related to cilia. The cilia in SSL is not polar.The P5 cells had nomal chromosome. Cells in ALL culture expressed FOXJ1 andβ-Tubulin.Conclusions The method of SSL culture and ALL culture could get the nasal epithelium cells. The cells from SSL culture can passage. The cells from ALL was helpful for cell cilia differentiation. Part three Ciliated Cells of Nasal Mucusa Differentiated from Human Umbilical Cord Blood Mesenchymal Stem CellsObjectives To investigate the differentiation of umbilical cord blood mesenchymal stem cells (UCB-MSCs) cultured in ALI, the transwell polycarbonate membrane insert coated by rat tail tendon collagen type I and the bronchial epithelial cell medium(BEpiCM) existed.Methods UCB-MSCs(P3) was transinfected by rAAv2-EGFP.The transfection efficiency was detected by flow cytometry after 48 hours. UCB-MSCs was cultured in the transwell polycarbonate membrane insert coated by rat tail tendon collagen type I,then ALL culture was created.The bronchial epitheliaol cell medium,which was beneficial to respiratory epithelial,was used. Cells were observed on inverted phase microscopy.The MSCs was collected after 1 and 2 week.Total RNA was prepared by using the RNeasy kit. The MUC8mRNA was detected . At the same time , FOXJ1 andβ-Tubulin were detected by immunofluorescence. Immunofluorescent staining of FOXJ1 andβ-Tubulin were observed on fluorescence microscope.Results The UCB-MSCs transinfected by rAAv2-EGFP expressed green fluorescein after 2 hours. The transfection efficiency after 48 hours was 97.9%. RT-PCR results showed that, UCB-MSCs did not express MUC8mRNA, which expressed in nasal epithelium. With the induction time, 1 week MUC8mRNA weak expression, enhanced after cultured for two weeks. Anti-β-Tubulin antibody was not detected cultured for two weeks. The positive FOXJ1 expression of red fluorescence can be observed in the context of UCB-MSCs marked green fluorescent protein. On confocal microscope in combination, as can be seen, red nucleus can be seen FOXJ1 expression of positive fluorescence in some of the MSCs marked green fluorescent.Conclusions UCB-MSCs was cultured on ALI,coated by rat tail tendon collagen type I and bronchial epithelial cell medium(BEpiCM) existed can be induced to expression the nasal ciliated cell marker MUC8 and ciliogenesis protein FOXJ1.UCB-MSCs maybe a new method to heal nasal diseases.